Infection of humans by Borrelia burgdorferi (Bb) can result in a multisystemic, chronic disease with diverse manifestations. We are investigating B. burgdorferi at several levels in order to understand some aspects of the disease pathogenesis. These studies include genetic manipulations of the spirochete and characterization of key structural and regulatory elements. 1. Molecular Characterization: Several features of Bb are being addressed. We have characterized homologous recombination between the genes encoding the major outer surface proteins (Osps) that both deletes osp gene sequences and creates chimeric gene fusions. Bb expressing recombinant osp genes can 'escape' killing by Osp-specific neutralizing antibodies. We are also investigating mechanisms of transcriptional regulation of osp gene expression. Isolating and studying genes whose expression is increased after a temperature upshift (heat shock genes) will provide information about their roles in cell growth, pathogenesis, plasmid replication and adaptation to stress. Understanding the mechanism of linear plasmid replication may yield a new target for antibiotics, in addition to shedding light on ways in which organisms manipulate their genetic material. 2. Gene Transfer: In order to allow genetic analysis of Bb, a method for gene transfer into Borrelia is being developed. A derivative of the Bb 16 kb linear plasmid will be used as a vector for electroporation, allowing for maintenance of transferred DNA in a native setting. DNA encoding recombinant Osp proteins that lack neutralizing epitopes will be introduced as a selectable marker. 3. cDNA Library: Extended passage of Bb in vitro eliminates its ability to infect rodents without any obvious differences in protein synthesis. cDNA libraries of high and low passage Bb are being constructed and clones derived from putative low abundance, differentially expressed messages will be identified by subtractive methods. A similar approach will be taken to identify messages that are differentially expressed in response to other biologically relevant stimuli.